In molecular imprinting in the presence of a template, polymers (MIPs) with high level of cross-linking are produced. The template corresponds regarding its structure and functionality to the target molecule [1, 2, 3, 4] and is after the synthesis removed, whereby cavities with forms and functionalities according to the template, remain. A number of imprinted polymers have until now been produced and the imprinting process constitutes a very promising way regarding the accomplishment of a large number of selective separations. The MIPs are distinguished by their high selectivity and affinity. In this way numerous materials with antibody like affinities can be produced. The advantages of the MIPs are their high association constants (Ka up to 108 M−1) for the target molecule [5, 6] and their high stability. Disadvantages are the limited chromatographic separation capacity (broad and asymmetrical peaks at liquid-chromatography, LC), low load capacities (<3 mg/g), as well as a complicated preparation process with low yield. The low yield prevents a scaling up and the use of more expensive templates. Besides, there are only a few examples of successful imprinting of biological macromolecules.
(i) Production of Polymers with Molecular Recognition Properties (MIPs) as Monodispersed Particles.
Most of the MIPs are produced in the presence of a template through free radical polymerization of functional, unsaturated monomers (vinyl-, acryl- methacryl-) and an excess of di- or tri-unsaturated monomers (vinyl-, acryl-, methacryl-) as cross-linkers, where by porous, organic networks are produced. This method has the advantage that relatively stable polymers can be produced with the use of different solvents and at different temperatures [7], which is important in view of the different solubilities of the various templates.
Most of the non covalent molecular imprinting systems are based on acryl- or methacrylmonomers, as for instance methacrylic acid (MAA), which is cross-linked with ethyleneglycoldimethacrylate (EDMA). For the production of imprinted stationary phases for chiral separation (chiral stationary phases, CSPs) at the beginning derivates of amino acid enantiomers were used as templates. This system can generally be used for the imprinting of templates via hydrogen bonding or electrostatic interaction with MAA [8, 9]. The method is explicitly demonstrated by the example of L-Phenylalaninanilid (L-PA).
In the first step the template (L-PA), the functional monomer (MAA) and the cross-linker (EDMA) are dissolved in a solvent with insignificant tendency towards forming hydrogen bonds and with small to average polarity. The free radical polymerization is thereafter started with an azo-initiator, for instance azo-N,N′-bis-isobutyronitril (AIBN), either photochemically at room temperature [11, 10] or thermochemically at 60° C. or higher [11]. The MIPs are formed as monoliths and before they are used they have to be crushed with mortar and pestle or with a ball mill. Following sieving the particles are sorted in the fractions 28-38 μm for chromatography resp. 150-200 μm for batch applications [11, 12, 13, 14, 15]. The template is extracted with a Soxhlet apparatus and thus recovered. The polymers as stationary phases are then evaluated by chromatography and the retention time and the capacity factors (k′) [16] of the templates are compared with those of analog structures.
The work up of the polymers by crushing and sieving is associated with high costs and and a high loss of material in the form of fine particles. Besides it is difficult to produce these materials on a larger scale. By the sieving of the monolith particles irregular particles arise which not only have surface localized binding sites but also binding sites with poor accessibility. Due to flow disturbances and diffusion limitations this causes a poor separation performance in chromatography [17].
Therefore there is a need to produce MIP-materials in large quantities and with homogeneous morphology, as these regarding their mass transfer properties and their load capacity are superior compared to the irregular particles from the monolith method. Materials with homogenous morphology are already produced by bead polymerization [12, 18], dispersion polymerization [13] or precipitation polymerization [14]. The morphology of these products are very sensitive to small changes regarding the synthesis conditions and besides, only certain solvents can be used for the polymerization. By consequence the synthesis for each target template has to be optimized which is costly and clearly limits the use of this synthesis variant. In addition, the synthesis conditions for the production of spherical particles are not always compatible with those synthesis conditions which lead to a higher selectivity and affinity for the template molecules. An alternative is the coating of preformed support materials [15, 19, 20], through which MIPs can be produced on metallic oxides. [15, 20] Another is the coating of the MIPs on organic polymer supports [19] or on the walls of fused silica capillaries [21, 22, 23].
For instance, for the production of molecular imprinting polymer coatings, wide-pore silica gels (Silica 60, Silica 100, Silica 500 or Silica 1000 (Merck)), modified with 3-(Trimethoxysilyl)propyl-methacrylate and in connection treated with Hexamethyl-disilazanes (end-capping) have been used. The support is then coated with a thin layer (10-156 Å) of a monomer mixture (ethylenglycoldimethacrylat, EGDMA and meth-acrylic acid, MAA) in the presence of a chiral template and an initiator (azo-bis(isobutyronitril), AIBN). After polymerization (monomer grafting approach) [15], the resulting silica gels were sieved by wind sieving and thereafter sedimented and tested chromatographically. An other method consists in the coating of LiChrosphere 1000 with a metal complexing polymer layer. This is performed by coating of propylmethacrylat derivatized silica particles with a metal complexing polymer in presence of a metal coordinating template [24].
(ii) Use of Non Porous Silica Particles as Pore Template and Production of Polymer Vesicles
The production of mesoporous polymers by use of colloidal silica particles as template was described by Johnson et al. [25]. Silica gel particles between 15 and 35 nm were settled and stabilized under pressure and heat. The spaces in the thus produced agglomerates of silica gel particles were then filled with a mixture of divinylbenzene (DVB) and an initiator (AIBN) and the polymerization started by heating to 60° C. The silica template was then dissolved with hydrofluoric acid or ammonium resp. cesium fluoride, whereby a vesical polymer frame was obtained, where the pore diameter can be varied through the dimension of the collodial silica gel.
Micro- and nanovesicles, which allows the inclusion of different materials, can be produced by spontaneous self-assembly of for instance amphiphilic block-copolymers [26, 27] or phosphorous lipids [28], by emulsion polymerization or by coating of colloidal particles with organic multi-layered films. Thus it is possible to stepwise coat collodial melamin resin particles with polyelectrolyte molecules and subsequently dissolve the core [29]. The weakly cross linked melamine resin particles with density in the size range 0,1 to 10 μm serve as template, which can be dissolved at pH-values <1,6. The template is repeatedly coated with alternately charged polyelectrolytes. This coating is also stable after having dissolved the template, whereby polyelectrolyte vesicles are obtained whose dimensions are determined by the dimension of the template. The strength of the wall is determined in advance by the coating and can within wide limits be freely determined. In such a way polyelectrolyte vesicles can be produced, which show a selective permeability for polymers depending on their molecular weight. [30].
The selective permeability can for instance be used in order to create ionic conditions inside the vesicles which differ significantly from the volume phase [31].
By the use of neutral templates of type CnH2n+1NH(CH2)2NH2 and tetraethoxysilane as silica source mesoporous molecular sieve with vesical structure can be obtained [32]. The vesicle is formed with one or more ca. 3-70 nm thick, wavy silica layers. The silica layers have mesopores in the size range 2,7-4,0 nm. The silica vesicles which are in the size range ca. 20 nm to 1400 nm, show a high thermal and hydrothermal stability.
(iii) Production of Non Porous Silica Gel Particles in the Range of Submicrometer and Micrometer
The synthesis of non porous silica gel particles in the range of submicrometer and micrometer up to ca. 4 μm is based on works of Stöber et al. [33], whereby by hydrolysis and condensation of tetraalkoxysilanes in ammonia water and with ethanol as cosolvent monodisperse, spherical silica gel particles up to 1,6 μm, resp. polydisperse particles up to 3 μm are formed. The synthesis is exhaustively investigated in several reports [34, 35, 36]. The production of monodisperse silica gel particles up to 2,0 μm by hydrolysis and condensation of tetraethoxysilan at −20° C. [37] and the synthesis of spherical silica gel particles under acidic conditions have been described [38]. The synthesis of larger non porous silica gel particles up to 10 μm is performed in the two-phase system alkoxysilan/water, where, due to the ethanol formed during the reaction, the system is slowly transformed to one single phase. [39].
(iv) Synthesis of Monodisperse, Spherical, Porous Silica Gel Particles in the Submicrometer and Micrometer Range
The production of porous silica gel particles in the range of micrometer (>5 μm) is performed by emulsion polymerization [40]. These exhibit a wide particle size range and have to be sieved. Porous silica gel particles in the nanometer range (<10 nm) have been described by Chu et al. [41]. The synthesis is based on a two-phase sol-gel process of silica without cosolvent.
Regarding the production of monodisperse, spherical, porous silica gel particles in the submicrometer range there is nothing known in the literature.
(v) Synthesis of Monodisperse, Spherical, Mesoporous Core/Shell Silica Gel Particles in the Submicrometer Range.
There is nothing known in the literature regarding synthesis of monodisperse, spherical silica gel particles with a non porous core and a mesoporous layer.